Transcription factors of the homeodomain superfamily play a major role in the development, differentiation and regulation of the nervous system. Members of the POU class III proteins (Brn-1, -2 and -4) are widely expressed during brain development but are restricted to specific neuronal cell groups in the adult hypothalamus, in particular, the magnocellular oxytocin (OT) and vasopressin (VP) expressing neurons of the supraoptic (SON) and paraventricular (PVN) nuclei, and of the parvocellular PVN. Ablation of the Brn-2 gene causes an inability of developing magnocellular neurons to migrate and differentiate into mature neurons. Consequently, homozygous Brn-2 (-/-) knockout mice lack OT- and VP-producing neurons as well as parvocellular PVN neurons, and have no posterior lobe of the pituitary glad. Heterozygous (+/-) Brn-2 knockout mice have a proportional reduction of OT and VP gene transcripts, indicating that Brn-2 expression levels in magnocellular neurons directly control the transcription of these neuroendocrine genes. The principal working hypothesis under investigation is that these recently cloned homeodomain proteins regulate OT and VP gene expression in the neuroendocrine hypothalamus. Proposed research is focused on the following specific aim: to determine the transcriptional action of the transfection. While the PI has utilized some molecular techniques in his research (i.e., single cell quantitative in situ hybridization histochemistry, immediate early gene mRNA induction, RNase protection assay and CNS antisense oligonucleotide microinfusion), he would like to become much more facile in molecular biology and its techniques in order to creatively pursue these reproductive hormone-neuroendocrine gene interactions at the level of gene expression. Proposed sabbatical work in the laboratory of Dr. Peter Burbach will focus on determining the transcriptional action of the brain homeodomain proteins (Brain-1, -2 and -4) on the oxytocin (OT) and vasopressin (VP) genes and identifying the cis-acting elements conferring the actions of these POU class III proteins using heterologous expression in tumor cell lines and homologous expression in organotypical cell cultures. This work will involve the design, construction and use of promoter-reporter gene constructs for the 5'- and 3'-flanking regions of the OT and VP genes, transfection by calcium-phosphate precipitation, biolistics, and or recombinant adenoviral infection in neuronal (Neuro2A cells) and non- neuronal tumor cell lines (293 human embryonal kidney cells, monkey kidney CV-1 cells, BHK cells and P19 EC cells), reporter gene assays (luciferase and beta-galactosidase), DNase I footprint analysis and electrophoretic mobility shift assay (EMSA) utilizing COS cells and in vitro translation. This experience should well position the PI to subsequently pursue an innovative cellular and molecular analysis of THE, GAD and GnRH gene regulation by these reproductive hormones in the neuroendocrine hypothalamus. Brain homeodomain proteins Brn-1, -2 and -4 on the OT and VP genes, and to identify the cis-acting elements conferring the actions of these POU class III proteins using heterologous expression in tumor cell lines and homologous expression in organotypical cell cultures. This research will utilize promoter- reporter gene constructs for the 5'- and 3'-flanking regions of the OT and VP genes, transient transfection of these constructs into neuronal and non-neuronal tumor cell lines and into organotypic cell cultures, DNase I footprint analysis and electrophoretic mobility shift assay (EMSA) utilizing COS cells and in vitro translation. This research will further our understanding of the role of these POU class II homeodomain proteins in the transcriptional regulation of OT and VP gene expression in the neuroendocrine hypothalamus.